Project Summary The long-term goal of our laboratory is to elucidate the molecular basis for intestinal homeostasis and its dysregulation in intestinal inflammation, and to develop novel approaches for prevention and therapy of inflammatory bowel diseases (IBD). The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and IBD. Autophagy (macroautophagy) is an intracellular degradation system that delivers unnecessary or dysfunctional cellular cargo sequestered inside double-membrane vesicles (autophagosomes) to the lysosome. Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. We will address our hypothesis with the specific aims of (1) To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier; (2) To elucidate the mechanistic role of intracellular signaling in autophagy regulation of intestinal TJ barrier; and (3) To delineate the protective role of autophagy-mediated enhancement of intestinal TJ barrier function in murine models of IBD. We will use innovative technical tools such as in vivo siRNA technology, in vivo full length mice colon perfusion, in vivo intestinal TJ protein trafficking, and high content quantitative imaging to study our specific aims. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.